Pistollato Francesca, Canovas-Jorda David, Zagoura Dimitra, Price Anna
Directorate F - Health, Consumers and Reference Materials, Joint Research Centre.
Directorate F - Health, Consumers and Reference Materials, Joint Research Centre;
J Vis Exp. 2017 Jun 9(124):55702. doi: 10.3791/55702.
Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.
人类多能干细胞可分化为多种细胞类型，可应用于基于人体的体外毒性试验。一个主要优点是，将体细胞重编程以产生人类诱导多能干细胞（hiPSC）避免了与使用人类胚胎干细胞（hESC）相关的伦理和法律问题。hiPSC可以扩增并有效分化为不同类型的神经元和神经胶质细胞，作为毒性测试的测试系统，特别是用于评估神经毒性中涉及的不同途径。这项工作描述了一种将hiPSC分化为神经元和神经胶质细胞混合培养物的方案。定义了由神经元分化调节和/或激活的信号通路。这些信息对于将细胞模型应用于新的毒性测试范式至关重要，在新范式中，根据化学物质干扰生物途径的能力对其进行评估。作为概念验证，使用线粒体呼吸复合物I的抑制剂鱼藤酮来评估Nrf2信号通路的激活，Nrf2信号通路是抗氧化反应元件（ARE）驱动的细胞抗氧化应激防御机制的关键调节因子。
